Mitochondria are subcellular organelles that play a central role in processes such as oxidative phosphorylation and apoptosis. Mitochondria contain several copies of a mitochondrial genome, the maintenance of which is vital for normal mitochondrial function. Mitocondrial DNA (mtDNA) is particularly sensitive to damage and mutation from a variety of endogenous and environmental insults. Mutations in mtDNA are responsible for several mitochondrial diseases, and are suspected to contribute to the etiology of other age-related disorders including neurodegenerative disorders, muscle-wasting, diabetes, and cancer. Given the observation that many cancer cells undergo changes in normal cellular metabolism, it is hypothesized that accumulated mtDNA mutations may be an important driving force in cancer development and/or progression. Although a strong association exists between cancer and mtDNA mutations, and certain mutations have been shown to increase metastatic potential, it has never been definitively demonstrated that mtDNA mutations cause cancer. The goal of this proposal is to determine if either mitochondrial dysfunction or mtDNA mutations can drive cancer formation. This will be accomplished under the direction of the following Specific Aims: (1) generate a systematic library of randomly generated, clonally expanded mitochondrial mutations; (2) screen the library for clones displaying a respiration deficiency; and (3) evaluate whether respiration deficiency and/or specific mitochondrial mutations precede and drive cancer development in human cells. The library will be prepared by treating normal human fibroblast cells to a variety of mitochondrial mutagenic agents, reversibly depleting the mitochondrial DNA, and isolating subsequent clones harboring clonally expanded mutations. Respiration competence will be assessed by measuring oxygen consumption and extracellular acidification rates. Tumorigenic potential will be measured using soft agar and matrigel mobility assays. Finally, generation of cybrid cell lines from select clones will determine if tumorigenic potentialis co- transferred with the mitochondria. The insights gained from this research will have important ramifications on our understanding of the role of mitochondrial mutations in the etiology and development of cancer. Once the precise role for mtDNA mutations is known, potential preventative and therapeutic strategies addressing the sources of accumulated mtDNA damage (i.e. diet and exposure to environmental mutagens) can be more efficiently developed. PUBLIC HEALTH RELEVANCE: Mitochondrial mutations and mitochondrial dysfunction are associated with cancer. However, it remains unclear if these are a cause or consequence of cancer development. Mitochondrial DNA may be a valuable therapeutic target once we understand the mechanisms of mtDNA mutagenesis and its relationship to cancer formation and progression.